| Background and objectiveIn recent years, people of our country enjoy a higher standard of living, but the incidence of colorectal cancer (CRC) increases gradually because of the westernizing of diet habit. Now, CRC has been the fourth most common cancer in our country. Its prognosis depends on the range involved. Five-year survival is 90% if CRC is diagnosed while still localized (ie, confined to the wall of the bowel), but only 68% for regional disease (ie, disease with lymph node involvement), and only 10% if distant metastases are present. So CRC screening in the average-risk population can reduce the incidence and mortality of CRC through the detection of early-stage adenocarcinomas and the detection and removal of adenomatous polyps. The common used tests in CRC screening includes fecal occult blood test (FOBT), double-contrast barium enema (DCBE), flexible sigmoidoscopy (FSIG), colonoscopy, and computed tomographic colonography (CTC). In the last decade, stool DNA test--a new noninvasive technology of CRC screening has been developed, which has high sensitivity and specificity, and requires no special bowel preparations. American cancer society (ACS) published the latest guidelines of screening and surveillance for the early detection of colorectal cancer and adenomatous polyps in March 2008. American College of Gastroenterology (ACG) also updated the guidelines for colorectal cancer screening in February 2009. Stool DNA test has been one of the recommended screening methods according to the two guidelines. The markers of stool DNA test includes 21 separate point mutations in the KRAS, APC, P53 genes; microsatellite instability; DNA methylation, and so on. To achieve adequate sensitivity, a multitarget DNA stool assay is required. But it is too expensive to be widely used. In order to reduce the costs of CRC screening, some foreign scholars have detected CRC with quantifying Human long DNA in stool, which has not been reported in our country. Therefore, we quantified Human long DNA in stool from 30 cases of colorectal cancer and 30 normal controls with fluorescent quantitative PCR to study the feasibility of this technology in CRC screening.Materials and Methods60 cases including 30 CRC and 30 normal controls were randomly selected from the patients in the first affiliated hospital of zhengzhou University from May to September,2009. All the diagnoses were verified by pathology. The average age of the 30 patients with CRC was 57.27±13.56 (range,21-82 yr), and 17 of the 30 patients were men. The ratio of male to female was 1.31:1. Among 30 cases of normal controls included 15 males and 15 females, and the ratio of male to female was 1:1. The oldest age is 79 years old while the youngest is 32 years old and the average age is 54.23±12.83 years old. Individuals were excluded if any of the following conditions applied:(1) personal history of, or coexistent cancer; (2) active therapy with chemotherapy or radiation therapy; (3) any invasive operation, such as colonoscopy or enema. Stool samples were collected before colonoscopy or surgery from the 60 patients. The samples were sent to the laboratory immediately and stored in a refrigerator set at-70℃. Total DNA was extracted from stool samples with QIAamp DNA Stool Mini kit (Qiagen, German). DNA was finally eluted in 200μl buffer AE and stored at-20℃.Primers specific for the human Alu sequences were used to amplify sequences-245bp inside Alu repeats, and the human long DNA in stool was quantified. Statistical tests were done using SPSS17.0 statistical software. Linear Regression Method was used to create Standard curve. x2 test was used to compare the sensitivities of the two screening methods. Wilcoxon signed-rank test was used to compare the human long DNA levels of different stool groups. Results1. Among 60 stool samples,59 cases of human long DNA was quantifiable.2. The long DNA was significantly higher in stools from CRC patients than from normal controls (median,84 vs.4.9ng/g stool; P=0.001). At a long DNA cutoff of 150ng/g stool, sensitivity for CRC was 46.67%, and specificity was 100%.3. The long DNA was significantly higher in distal CRC stools than from proximal CRC (281.5ng/g vs.7.52ng/g; P=0.008);and was not influenced by gender, age, tumor size, or Dukes stage.4. The sensitivity of long DNA for CRC was significantly higher than FOBT(46.67% vs.26.67%, P=0.031)Conclusions1. Fluorescent quantitative Alu PCR is a simple method to quantify human long DNA in stool.2. The long DNA in stool was influenced by tumor site, and was independent of gender, age, tumor size, or Dukes stage.3. The sensitivity of long DNA for CRC was significantly higher than FOBT.4. Quantifying human long DNA in stool with fluorescent quantitative Alu PCR has the advantage of lower cost compared with other approaches to assay long human DNA in stool. |
